Genes Associated With Post Relapse Survival And Uses Thereof

ABSTRACT

Provided are methods, systems and kits for predicting post-relapse survival of a cancer patient and for identifying cancer genes predictive of the post-relapse survival of the patient. Values representing gene expression levels of a group of genes associated with survival of the cancer cells are determined using gene expression profiling platforms and a plurality of probe sets that hybridize to one or more of the genes in the group. A predictive model establishes a predictive value based on the weighted contribution of each gene associated with survival of the cancer cells to risk of death for the cancer patient and imports expression values of the genes in the group that is indicative of a risk of death for the relapsed patient. Using global gene expression profiling and statistical analysis, expression of cancer cell genes at baseline and at first relapse that are involved in interaction of cancer cells with cells in their microenvironment, can be used to identify genes that are predictive of post-relapse survival.

RELATED APPLICATION(S)

This application is a continuation-in-part and claims priority to Ser. No. 13/068,008, filed 29 Apr. 2011.

The entire teachings of the above application(s) are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grants CA-113992, CA-093897 and CA-055819 awarded by the National Cancer Institute. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of gene expression profiling and cancer prognosis. More specifically, the present invention discloses methods and systems for a predictive model utilizing a group of genes associated with survival of cancer cells to predict post-relapse survival of a cancer patient.

2. Background Description

Multiple myeloma is unique among the hematological malignancies in that in the vast majority of patients its growth is restricted to the bone marrow. Development of myeloma is intimately associated with osteolytic bone disease in over 80% of patients, as a result of inhibition of osteoblast differentiation and stimulation of osteoclastogenesis. Myeloma is also unique among all tumors that metastasize to the bone marrow and cause osteolysis; myeloma-induced osteolytic lesions do not repair, even after many years of complete remission. Myeloma associated lytic bone disease results from disruption of the RANKL/OPG axis, an effect likely mediated by myeloma-cell secretion of the Wnt signaling inhibitor Dickkopf-1 (DKK-1). By inhibiting Wnt signaling, DKK-1 blocks the differentiation of bone marrow mesenchymal cells (MSC) to osteoblasts, increasing expression of RANKL and reducing expression of OPG, resulting in stimulation of osteoclast formation and activity. Indeed, in myeloma patients the soluble RANKL/OPG ratios correlate with the extent of osteolytic bone disease.

Progression of the pre-malignant plasma cell dyscrasia monoclonal gammopathy of unknown significance (MGUS) to myeloma is preceded by changes in bone turnover rates; an initial coupled increase in both osteoblast and osteoclast activity is followed with disease progression by decreased osteoblast activity while osteoclast activity remains elevated, leading to osteolytic bone disease. Myeloma cell dependence on the bone marrow microenvironment and on the changes they induce in the bone marrow is also evident in a SCID-hu model for primary human myeloma, where growth of freshly obtained primary myeloma cells is restricted to the human bone implants. Using this model, it was demonstrated that myeloma growth is dependent on osteoclast activity. This observation was reproduced in culture, where osteoclasts supported myeloma cells survival.

In contrast to osteoclasts, which always support myeloma cell survival in vitro and in vivo, osteoblast effects in the SCID-hu model varied from none to increase in bone formation associated with inhibition of myeloma growth. In co-cultures, osteoblasts differentiated from mesenchymal cells inhibited survival of freshly isolated myeloma cells, suggesting that inhibition of osteoblast differentiation supports myeloma cell survival. The interactions between myeloma cells and bone cells, as well as the molecular consequences of myeloma cell interactions with osteoclasts and mesenchymal cells are not well understood.

While new myeloma therapies have achieved high rates of complete remission and near-complete remission (1), relapses are common, and most patients experience short post-relapse survival, Thus, there is a recognized need in the art to better able to predict the likelihood of survival of a myeloma patients after relapse of the cancer. The prior art is deficient in the identification of genes that are associated with the survival of myeloma cells and are potential targets for interventions, and methods and systems for predicting post relapse survival of myeloma patients. The present invention fulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

The present invention is directed to a method for predicting post-relapse survival of a cancer patient in a state of relapse. The method comprises importing individual values for gene expression of a group of genes associated with survival of cancer cells obtained from the cancer patient after relapse of the cancer into a predictive model, which is a statistical model. Using the predictive model, a predictive value, based on the weighted contribution of each gene to a risk of death for the cancer patient and the imported expression values of the genes in the group, is established that is indicative of a risk of death for the relapsed cancer patient, thereby predicting post-relapse survival of the cancer patient.

The present invention is directed to a related method for predicting post-relapse survival of a multiple myeloma patient in a state of relapse. The method comprises hybridizing nucleic acids obtained from multiple myeloma cells in the relapsed patient to one or more platforms comprising probe sets hybridizable to one or more genes in a group of genes associated with survival of the multiple myeloma cells and converting intensity of a signal generated upon hybridization to the value of gene expression for each gene in the group. Values for gene expression of each gene in the group are imported into a predictive model, which is a statistical model. Using the predictive model a predictive value, based on the weighted contribution of each gene to risk of death for the relapsed multiple myeloma patient and the imported expression values of the genes in the group, is established that is indicative of a risk of death for the relapsed patient, thereby predicting post-relapse survival of the cancer patient.

The present invention also is directed to another method for predicting post-relapse survival of a cancer patient in a state of relapse. The method comprises measuring the level of gene expression of a group of multiple myeloma genes comprising at least CCNE2, PECAM1, HMOX1, and CISH; optionally further comprising HBEGF, JUN, SIX5, and DUSP1; optionally further comprising BMP6, FOSB, and LIME1; optionally further comprising multiple myeloma genes BIRC3, FER1L4, KLHL21, MAFF, SOCS3, and TSC22D3 from multiple myeloma cells obtained from the patient before the start of a treatment regimen for the cancer, and measuring the level of gene expression of the genes obtained from the patient after relapse of the multiple myeloma. The expression level of each gene before treatment is compared with the expression level of each corresponding gene after relapse where a decrease in expression of PECAM1, HMOX1, CISH, SIX5, BMP6, JUN, FOSB and DUSP1, and an increase in expression of LIME1, CCNE2, HBEGF has a statistically significant correlation with post-relapse survival of the myeloma cells in the patient and is predictive of a low likelihood of survival of the patient. The present invention is directed to a related method where the group of myeloma genes further comprises. BIRC3, FER1L4, TSC22D3, MAFF, SOCS3, and KLHL21. A decrease in expression level of BIRC3, FER1L4, and TSC22D3, and an increase in expression level of MAFF, SOCS3 and KLHL21 are predictors of a likelihood of a shorter survival of the patient.

The present invention is directed further to a system for predicting post-relapse survival of a multiple myeloma patient in a state of relapse. The system comprises one or more platforms having probe sets hybridizable to one or more multiple myeloma genes in a group comprising at least CCNE2, PECAM1, HMOX1, and CISH; optionally further comprising HBEGF, JUN, SIX5, and DUSP1; optionally further comprising BMP6, FOSB, and LIME1; optionally further comprising multiple myeloma genes BIRC3, FER1L4, KLHL21, MAFF, SOCS3, and TSC22D3 and a signal processor configured to convert intensity of a hybridization signal to a value of gene expression for each gene in the group. A predictive model configured to import the gene expression values comprises a calculator that uses a summation function of an assigned risk of death for each gene in the group to calculate the risk of death of the relapsed patient, where risk for each gene is assigned on a sliding scale and is a product of each gene's weight in determining risk of death and the imported expression value of the gene.

The present invention is directed further still to a kit for predicting post-relapse survival of a multiple myeloma patient in a state of relapse. The kit comprises the predictive model of the system and is tangibly stored on a computer storage medium. The present invention is directed to a related kit further comprising a platform that has a plurality of probes hybridizable to one or more of multiple myeloma genes CCNE2, PECAM1, HMOX1, and CISH; optionally further comprising HBEGF, JUN, SIX5, and DUSP1; optionally further comprising BMP6, FOSB, and LIME1. The present invention is directed to another related kit that further comprises a plurality of probes hybridizable to one or more of multiple myeloma genes BIRC3, FER1L4, TSC22D3, MAFF, SOCS3 and KLHL21.

The present invention is directed further still to a method for identifying cancer genes predictive of post-relapse survival for a cancer patient. The method comprises co-culturing cancer cells with cells that interact with the cancer cells in their microenvironment and performing a first global gene expression profiling on the cancer cells before co-culture, and a second global gene expression profiling after co-culture. From comparing the first and the second gene expression profile, a set of genes differentially expressed after co-culture are identified via statistical analysis. A third global gene expression profiling is performed on post-relapse cancer cells obtained from relapsed cancer patients and post-relapse genes whose expression was differentially changed are identified via statistical analysis of the third expression profile. A comparison between post-relapse expression of the genes whose expression differentially changed after co-culture and duration of survival of the post-relapse cancer patients identifies the cancer genes predictive of post-relapse survival of the cancer patient. The present invention is directed to a related method further comprising performing a multivariate permutation test to eliminate genes with a higher than a pre-determined false positive change in expression. The present invention is directed to another related method further comprising identifying networks of interrelated genes among the differentially expressed gene set to further narrow the genes comprising the same.

Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.

BRIEF DESCRIPTION OF DRAWINGS

So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions and certain embodiments of the invention briefly summarized above are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

FIG. 1 shows purity of myeloma cells recovered from co-culture with osteoclasts. Myeloma plasma cells were recovered from co-culture. The recovered cells were reacted with monoclonal antibodies to CD38 (PE conjugated) and CD45 (FITC conjugated) and analyzed by flow cytometry. The purity was routinely >95%.

FIGS. 2A-2E are five networks depicting interrelationships among 54 multiple myeloma plasma cell genes with high probability IPA scores of the 58 genes whose gene expression changes following co-culture with mesenchymal stem cells was similar to that following co-culture with osteoclasts.

FIGS. 3A-3C show a Kaplan-Meyer analysis of post relapse survival of patients. 127 patients that were treated with total therapy 2 were used as a training set, from which the predictive model was developed, where expression 11 genes, represented by 13 probe sets in Table 6 (FIG. 3A) predicted survival of the patients. The 11 genes in Table 6 predicted survival of 32 patients who relapsed on total therapy 3 protocol (FIG. 3B) and 98 patients who relapsed after various treatment protocols (FIG. 3C). Risk was assigned by BRB ArrayTools software using expression signal of 72 probesets identified by co-culture experiments. Expression signals were dichotomized at the median.

FIGS. 4A-4D are bar graphs summarizing the correlation between microarray-based gene expression profiling (GEP; FIGS. 4A and 4C) and quantitative real-time polymerase chain reaction (qRT-PCR; FIGS. 4B and 4D) for PECAM (FIGS. 4A and 4B) and CCNE2 (FIGS. 4C and 4D) in 10 patients. The mRNA used in both the GEP and qRT-PCR was from a single preparation from isolated plasma cells.

FIGS. 5A-5C show a Kaplan-Meyer analysis of post relapse survival of patients evaluated by the 33 probeset model in Table 7. 127 patients were subject to Total Therapy II (TT2, the training set; FIG. 5A), 98 patients were treated by various protocols (FIG. 5B), and 32 patients were treated by Total Therapy III (TT3; FIG. 5C).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art.

As used herein, the term, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein “another” or “other” may mean at least a second or more of the same or different claim element or components thereof. The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included.

As used herein, the term “or” in the claims refers to “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or”.

As used herein, “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term “about” generally refers to a range of numerical values (e.g., +/−5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term “about” may include numerical values that are rounded to the nearest significant figure.

As used herein, the term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. The activity of such agents may render it suitable as a “therapeutic agent” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.

A “patient,” “individual,” “subject” or “host” refers to either a human or a non-human animal, e.g., non-human mammals. The term “mammal” is known in the art, and exemplary mammals include humans, primates, bovines, porcines, canines, felines, and rodents, e.g., mice and rats.

The invention provides, inter alia, methods, systems, and kits for predicting post-relapse survival of a cancer patient, particularly a multiple myeloma patient, using groups of multiple myeloma genes. In one embodiment, the group of multiple myeloma genes consists of, consists essentially of, or comprises CCNE2, PECAM1, HMOX1, and CISH. In certain embodiments, the group of multiple myeloma genes further comprises HBEGF, JUN, SIX5, and DUSP1 (i.e. the group consists of, consists essentially of, or comprises CCNE2, PECAM1, HMOX1, CISH, HBEGF, JUN, SIX5, and DUSP1). In more particular embodiments, the group of multiple myeloma genes further comprises BMP6, FOSB, and LIME1 (i.e. the group consists of, consists essentially of, or comprises CCNE2, PECAM1, HMOX1, CISH, HBEGF, JUN, SIX5, DUSP1, BMP6, FOSB, and LIME1). In still more particular embodiments, the group of multiple myeloma genes further comprises BIRC3, FER1L4, TSC22D3, MAFF, SOCS3, and KLHL21 (i.e., the group consists of, consists essentially of, or comprises CCNE2, PECAM1, HMOX1, CISH, HBEGF, JUN, SIX5, DUSP1, BMP6, FOSB, and LIME1, BIRC3, FER1L4, TSC22D3, MAFF, SOCS3, and KLHL21). In other embodiments, the group of multiple myeloma genes consists of, consists essentially of, or comprises the genes in Table 7 (i.e. OSBPL10, CCNE2, UBE2D1, PECAM1, APOE, ATP6AP2, PTPRG, ZNF267, PECAM1, HMOX1, SOD2, TXNRD1, CD58, MS4A7, PPBP, EPAS1, FLNA, EPHB1, PLA2G7, CISH, SQSTM1, KLHL14, ATP6V1C1, FAM13A, PECAM1, SCD5, GM2A, CREB3L2, CD163, CPM, IDUA, SPAG4, and Clorf38—optionally, e.g., using the particular probes recited in Table 7). In still other embodiments, the group of multiple myeloma genes consists of, consists essentially of, or comprises the genes in Table 2 (i.e. AMPD 1, ANPEP, BIRC3, BMP6, BTLA, CBFA2T3, CCNE2, CCR2, CCR7, CD44, CD48, CD82, CEP170, CHST11, CISH, CITED2, DUSP1, EPB41L3, FAM129B, FER1L4, FOS, FOSB, GLA, GPRC5D, HBEGF, HMOX1, ICAM1, IFI30, IL8, ISG20, JUN, KANK1, KLF2, KLHL21, LIME1, LRRN1, MAFF, MMP14, MT1E, MT1H, PECAM1, PLAC8, PLAU, PLAUR, PRRG4, SAT1, SGK1, SIX5, SLC25A19, SLC2A4RG, SOCS3, SPHK1, TGM2, TMEM49, TNFAIP2, TNFRSF17, TSC22D3, and UGT2B17—optionally, e.g., using the particular probes recited in Table 2).

Any of the following methods, system, or kits may be readily adapted to use the different groups of multiple myeloma genes above. The predictive values of the genes can be evaluated in a variety of ways, e.g., according to the hazard ratios in any of Tables 4 or 5 (e.g., for genes with a hazard ratio>1, increased expression correlates with low likelihood of survival and genes with a hazard ratio<1, increased expression correlates with high survival) or the weights in Table 7 (positive values indicate genes where an increase in expression is associated with low survival, negative values indicate genes where an increase in expression is associated with high survival). Expression levels are evaluated by any suitable means, such as PCR, microarray, or sequencing, including combinations of these. In particular embodiments, the levels are measured using microarray, such as oligonucleotide microarrays, including AFFYMETRIX® arrays, such as U133Plus2.0 microarrays. In still more particular embodiments, expression levels are evaluated on an oligonucleotide microarray using the probes provided in Tables 1-7. Levels are typically measured in multiple myeloma plasma cells. In particular embodiments, the multiple myeloma plasma cells are CD138+. The cells are isolated at any time, but in particular embodiments are isolated at about the time of first relapse, e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of the first clinical signs of relapse, or in some embodiments, within about 1, 2, 3, 4, or 5 weeks, or within about 1, 2, 3, 4, 5, or 6 months of the first clinical signs of relapse.

In one embodiment of the present invention there is provided a method for predicting post-relapse survival of a relapsed cancer patient (e.g., a cancer patient in a state of relapse), comprising importing individual values for gene expression of a group of genes associated with survival of cancer cells obtained from the cancer patient after relapse of the cancer into a predictive model, where the predictive model is a statistical model; and establishing, with the predictive model, a predictive value based on the weighted contribution of each gene to risk of death for the cancer patient and the imported expression values of the genes in the group that is indicative of a risk of death for the relapsed cancer patient, thereby predicting post-relapse survival of the cancer patient.

In this embodiment obtaining values for gene expression of the genes in the group may comprise hybridizing nucleic acids obtained from the cancer cells under standard conditions to one or more platforms comprising probe sets hybridizable to one or more genes in the group; and converting intensity of a signal generated upon hybridization to the value of gene expression for each gene in the group. Also, in this embodiment establishing the predictive value may comprise summing the products of the weighted risk of each gene in the group in the predictive model and the imported expression level for each gene in the group. Weighted risk comprises a coefficient for each gene in the group where the coefficient is representative of each gene's weight contribution to a risk of death based on a hazard ratio for a 2-fold increase in gene expression such that, if the hazard is higher than 1, increased expression correlates to a higher risk of death and, if the hazard ratio is lower than 1, increased expression indicates a lower risk of death.

In an aspect of this embodiment the genes in the group of multiple myeloma genes consist of, consist essentially of, or comprise CCNE2, PECAM1, HMOX1, and CISH; optionally further comprising HBEGF, JUN, SIX5, and DUSP1; optionally further comprising BMP6, FOSB, and LIME1. Further to this aspect the genes in the group further comprises multiple myeloma genes BIRC3, FER1L4, KLHL21, MAFF, SOCS3, and TSC22D3. In all aspects of this embodiment the cancer may be multiple myeloma. In another related aspect, the genes in the group of multiple myeloma genes consists of, consists essentially of, or comprises multiple myeloma genes OSBPL10, CCNE2, UBE2D1, PECAM1, APOE, ATP6AP2, PTPRG, ZNF267, PECAM1, HMOX1, SOD2, TXNRD1, CD58, MS4A7, PPBP, EPAS1, FLNA, EPHB1, PLA2G7, CISH, SQSTM1, KLHL14, ATP6V1C1, FAM13A, PECAM1, SCD5, GM2A, CREB3L2, CD163, CPM, IDUA, SPAG4, and Clorf38. In yet another related aspect, the group of multiple myeloma genes consists of, consists essentially of, or comprises multiple myeloma genes AMPD1, ANPEP, BIRC3, BMP6, BTLA, CBFA2T3, CCNE2, CCR2, CCR7, CD44, CD48, CD82, CEP170, CHST11, CISH, CITED2, DUSP1, EPB41L3, FAM129B, FER1L4, FOS, FOSB, GLA, GPRC5D, HBEGF, HMOX1, ICAM1, IFI30, IL8, ISG20, JUN, KANK1, KLF2, KLHL21, LIME1, LRRN1, MAFF, MMP14, MT1E, MT1H, PECAM1, PLAC8, PLAU, PLAUR, PRRG4, SAT1, SGK1, SIX5, SLC25A19, SLC2A4RG, SOCS3, SPHK1, TGM2, TMEM49, TNFAIP2, TNFRSF17, TSC22D3, and UGT2B1.

In a related embodiment there is provided a method for predicting post-relapse survival of a relapsed multiple myeloma patient, comprising hybridizing nucleic acids obtained from multiple myeloma cells in the relapsed patient to one or more platforms comprising probe sets hybridizable to one or more genes in a group of genes associated with survival of the multiple myeloma cells; converting intensity of a signal generated upon hybridization to the value of gene expression for each gene in the group; importing values for gene expression of each gene in the group into a predictive model, where the predictive model is a statistical model; and establishing, with the predictive model, a predictive value based on the weighted contribution of each gene to risk of death for the relapsed multiple myeloma patient and the imported expression values of the genes in the group that is indicative of a risk of death for the relapsed patient, thereby predicting post-relapse survival of the cancer patient. In this embodiment the steps for establishing the predictive value, the weighted risk and the myeloma genes are generally described supra.

In another embodiment of the present invention there is provided a method for predicting post-relapse survival of a relapsed multiple myeloma patient, comprising measuring the level of gene expression of a group of multiple myeloma genes consisting of, consisting essentially of, or comprising at least CCNE2, PECAM1, HMOX1, and CISH; optionally further comprising HBEGF, JUN, SIX5, and DUSP1; optionally further comprising BMP6, FOSB, and LIME1 from multiple myeloma cells obtained from the patient before start of a treatment regimen for the cancer; measuring the level of gene expression of the genes obtained from the patient after relapse of the multiple myeloma; and comparing the expression level of each gene before treatment with the expression level of each corresponding gene after relapse; wherein a decrease in expression of PECAM1, HMOX1, CISH, SIX5, BMP6, JUN, FOSB, and DUSP1 and an increase in expression of LIME1, CCNE2, and HBEGF has a statistically significant correlation with post-relapse survival of the myeloma cells in the patient and is predictive of a low survival rate of the patient.

Further to this embodiment the group of multiple myeloma genes may comprise additional genes BIRC3, FER1L4, TSC22D3, MAFF, SOCS3, and KLHL21 and where decrease in expression level of BIRC3, FER1L4, and TSC22D3, and an increase in expression level of MAFF, SOCS3 and KLHL21 are predictors of shorter survival of the patient. In both embodiments measuring a level of gene expression of the genes in the group comprises hybridizing nucleic acids obtained from the myeloma cells to one or more platforms comprising probe sets hybridizable to one or more genes in the group; and converting intensity of a signal generated upon hybridization to the value of gene expression for each myeloma gene in the group.

In yet another embodiment the present invention provides a system for predicting post-relapse survival of a multiple myeloma patient in a state of relapse, comprising one or more platforms having probe sets hybridizable to one or more multiple myeloma genes in a group consisting of, consisting essentially of, or comprising at least CCNE2, PECAM1, HMOX1, and CISH; optionally further comprising HBEGF, JUN, SIX5, and DUSP1; optionally further comprising BMP6, FOSB, and LIME1; a signal processor configured to convert intensity of a hybridization signal to a value of gene expression for each gene in the group; and a predictive model configured to import the gene expression values and comprising a calculator that uses a summation function of an assigned risk of death for each gene in the group to calculate the risk of death of the relapsed patient, wherein risk for each gene is assigned on a sliding scale and is a product of each gene's weight in determining risk of death and the imported expression value of the gene.

Further to this embodiment the group of multiple myeloma genes may comprise additional genes BIRC3, FER1L4, TSC22D3, MAFF, SOCS3, and KLHL21. In both embodiments the predictive model comprises a computer program product tangibly stored in a computer memory or computer storage medium and configured to be executed by a processor. The predictive model comprises a coefficient, as described supra. In another related aspect, the probe sets are hybridizable to a group of multiple myeloma genes consists of, consists essentially of, or comprises multiple myeloma genes OSBPL10, CCNE2, UBE2D1, PECAM1, APOE, ATP6AP2, PTPRG, ZNF267, PECAM1, HMOX1, SOD2, TXNRD1, CD58, MS4A7, PPBP, EPAS1, FLNA, EPHB1, PLA2G7, CISH, SQSTM1, KLHL14, ATP6V1C1, FAM13A, PECAM1, SCD5, GM2A, CREB3L2, CD163, CPM, IDUA, SPAG4, and Clorf38. In yet another related aspect, the probe sets are hybridizable to a group of multiple myeloma genes consists of, consists essentially of, or comprises multiple myeloma genes AMPD1, ANPEP, BIRC3, BMP6, BTLA, CBFA2T3, CCNE2, CCR2, CCR7, CD44, CD48, CD82, CEP170, CHST11, CISH, CITED2, DUSP1, EPB41L3, FAM129B, FER1L4, FOS, FOSB, GLA, GPRC5D, HBEGF, HMOX1, ICAM1, IFI30, IL8, ISG20, JUN, KANK1, KLF2, KLHL21, LIME1, LRRN1, MAFF, MMP14, MT1E, MT1H, PECAM1, PLAC8, PLAU, PLAUR, PRRG4, SAT1, SGK1, SIX5, SLC25A19, SLC2A4RG, SOCS3, SPHK1, TGM2, TMEM49, TNFAIP2, TNFRSF17, TSC22D3, and UGT2B1.

In yet another embodiment the present invention provides a kit for predicting post-relapse survival of a multiple myeloma patient in a state of relapse, comprising the predictive model, as described supra, tangibly stored on a computer storage medium. Further to this embodiment, the kit comprises a platform having a plurality of probes hybridizable to a group of multiple myeloma genes consisting of, consisting essentially of, or comprising CCNE2, PECAM1, HMOX1, and CISH; optionally further comprising HBEGF, JUN, SIX5, and DUSP1; optionally further comprising BMP6, FOSB, and LIME1; optionally further comprising multiple myeloma genes BIRC3, FER1L4, KLHL21, MAFF, SOCS3, and TSC22D3. Further still, the kit may comprise a platform further having a plurality of probes hybridizable to one or more of multiple myeloma genes BIRC3, FER1L4, TSC22D3, MAFF, SOCS3 and KLHL21. In another related aspect, the probes are hybridizable to a group of multiple myeloma genes consisting of, consisting essentially of, or comprising multiple myeloma genes OSBPL10, CCNE2, UBE2D1, PECAM1, APOE, ATP6AP2, PTPRG, ZNF267, PECAM1, HMOX1, SOD2, TXNRD1, CD58, MS4A7, PPBP, EPAS1, FLNA, EPHB1, PLA2G7, CISH, SQSTM1, KLHL14, ATP6V1C1, FAM13A, PECAM1, SCD5, GM2A, CREB3L2, CD163, CPM, IDUA, SPAG4, and Clorf38. In yet another related aspect, the probes are hybridizable to a group of multiple myeloma genes consisting of, consisting essentially of, or comprising multiple myeloma genes AMPD1, ANPEP, BIRC3, BMP6, BTLA, CBFA2T3, CCNE2, CCR2, CCR7, CD44, CD48, CD82, CEP170, CHST11, CISH, CITED2, DUSP1, EPB41L3, FAM129B, FER1L4, FOS, FOSB, GLA, GPRC5D, HBEGF, HMOX1, ICAM1, IFI30, IL8, ISG20, JUN, KANK1, KLF2, KLHL21, LIME1, LRRN1, MAFF, MMP14, MT1E, MT1H, PECAM1, PLAC8, PLAU, PLAUR, PRRG4, SAT1, SGK1, SIX5, SLC25A19, SLC2A4RG, SOCS3SPHK1, TGM2, TMEM49, TNFAIP2, TNFRSF17, TSC22D3, and UGT2B1.

In yet another embodiment the present invention provides a method for identifying cancer genes predictive of post-relapse survival for a cancer patient, comprising co-culturing cancer cells with cells that interact with the cancer cells in their microenvironment; performing a first global gene expression profiling on the cancer cells before co-culture; performing a second global gene expression profiling on the cancer cells after co-culture; identifying via statistical analysis, from the first and second gene expression profile, a set of genes differentially expressed after co-culture; performing a third global gene expression profiling on post-relapse cancer cells obtained from relapsed cancer patients; identifying, via statistical analysis, from the third expression profile, those post-relapse genes that are also differentially expressed; and comparing post-relapse expression of these genes with duration of survival of the post-relapse cancer patients, thereby identifying the cancer genes predictive of post-relapse survival of the cancer patient. In one embodiment, the cancer cells are co-cultured with osteoclast cells. In another embodiment, the cancer cells are co-cultured with mesenchymal stem cells.

In a further embodiment the method may comprise performing a multivariate permutation test to eliminate genes with a higher than a pre-determined false positive rate of prediction. In another further embodiment the method may comprise identifying networks of interrelated genes among the differentially expressed gene set to further narrow the genes comprising the same. In all embodiments the ratio of change in expression may be a ratio of a change in signal intensity at relapse to a change in signal intensity at baseline. In an aspect of these embodiments the cancer cells may be multiple myeloma cells and the co-cultured cells are osteoclasts or mesenchymal stem cells.

Provided herein are methods, systems and kits utilizing a predictive model to predict post-survival relapse of a cancer patient, preferably, but not limited to, an individual with multiple myeloma. The predictive model is based on a group of genes that are shown statistically to beneficially affect the survival of myeloma cells after exposure to chemotherapeutic agents during a therapeutic regimen undergone by the patient. It is recognized that the global expression profiling techniques described herein are well-suited to identify genes associated with survival of other cancer cells and, as such, applicable predictive models can be constructed as predictive tools for calculating a risk of death for a cancer patient in which the cancer has relapsed. Platforms, such as DNA microarrays or RT-PCR arrays, measure gene expression levels and/or quantify signal intensity related to gene expression.

The predictive model provided herein is constructed utilizing gene expression values, i.e., levels, of the survival associated genes in relapse, such as the genes identified in Table 5 with the exception of PLAUR or, more preferably, the genes identified in Table 6, after the cancer patient has relapsed. A coefficient representing the weight contribution of each of the genes in promoting myeloma cell survival is determined based on the baseline gene expression values. This represents a sliding scale for assigning risk of death of the patient after relapse of the cancer. The predictive model comprises a calculator configured to utilize a summation function to calculate and assign risk. Risk is assigned based on the summation of products of the coefficient for each gene and the gene expression value of the gene after relapse.

The predictive model may be provided in a computer or other electronic device having one or more wired or wireless network connections, a memory to store the model and a processor to execute instructions enabling the predictive model on the computer or other electronic device. Such computers and electronic devices are well-known and standard in the art. The predictive model may comprise a computer program product tangibly stored in a memory on a computer or other computer storage device as are known in the art.

In constructing the predictive model provided herein, it is now widely accepted that the changes myeloma induces in the bone marrow microenvironment that result in osteolytic bone disease are not just manifestation. The cellular changes induced by myeloma cells supply factors and signals essential for the sustenance and progression of the disease. Co-cultures of myeloma cells with osteoclasts and with bone marrow mesenchymal stem cells are used to identify changes in gene expression by myeloma cells induced following these co-culture.

It is reasonable that genes required for myeloma cell survival will be among the genes whose expression similarly changes in both co-culture systems, such genes were selected for further study. It is interesting that from changes in the expression of over a thousand probe sets, only 72, corresponding to 58 genes, were common to both co-culture systems, indicating that the majority of the other observed changes were unique to the interaction of myeloma cells with osteoclasts or mesenchymal stem cells, and probably not associated with myeloma cell survival.

Changes in gene expression associated with myeloma cells survival are utilized as prognostic indicators. Expression of 22 of 58 genes changed in patients at relapse compared with baseline expression, and 7 genes (8 probesets) (PECAM1, ANPEP, PLAU (211668_S_AT), DUSP1, CCNE2, KLHL21, ICAM1 of these changes were significantly (p<0.05) associated with post relapse survival of myeloma cells. The genes associated with longer survival are:

1. Lower expression of the Wnt target regulator of cell cycle (CCNE2/Cyclin E2 (2);

2. Lower expression of KLHL21, which is a gene required for efficient chromosome alignment and cytokinesis (3);

3. Higher expression of the CD38 ligand PECAM1 (CD31), which is expressed on bone marrow myeloma cells, but not on extramedullary cells (4), targets cells for apoptosis and, together with cadherin 5 and β-catenin, is essential for angiogenesis (5);

4. Lower expression of ICAM-1 (CD54) whose expression is associated with cell adhesion mediated drug resistance (6) and is important for transendothelial migration (7);

5. Lower expression of PLAU 211668_S_AT (urokinase-type plasmin activator), a proteolytic enzyme that breaks down matrix and promotes invasion (8).

6. Higher expression of ANPEP (CD13) which is a protease present in soluble form in the plasma (9) and is involved in metabolism of regulatory peptides (10), is involved in tumor angiogenesis (111) and reduces availability of certain peptides to dendritic cells (9); and

7. Higher expression of DUSP1, the dual specificity phosphatase, a potential target of β-catenin that dephosphorylates Erks, JunK, and p38 MAPK and regulates the innate immune response (12).

While changes in gene expression at relapse point to emergence of more aggressive myeloma cells either by selection of pre-existing or new adaptations, they also mask the absolute level of expression, which by itself could be an important disease feature. Indeed, expression of 18 genes at relapse was significantly (p<0.05) associated with survival after relapse, some with high levels of significance.

In addition to the genes discussed above, other genes whose expression level is associated with longer survival of myeloma cells are:

8. Higher expression of components of the transcription regulator AP-1 JUN (13);

9. Higher expression of FOSB;

10. Higher expression of TSC22D3 (GILZ) which is a suppressor of AP-1 and NF-κB DNA binding activity (14);

11. Higher expression of HMOX1, the stress response heme oxigenase 1, which is upregulated in myeloma by oxidative stress (15-18); and

12. Lower expression of MAFF, a regulator of stress response and pro inflammatory cytokines, that is essential for antioxidant response element dependent genes and must cooperate with Nrf2 to elicit this response (19-21);

13. Higher expression of CISH, a member of the SOCS family which attenuate pro inflammatory signaling (22);

14. Higher expression of SIX5 which is expressed at low levels in many tissues, with known function in early development (23-26);

15. Higher expression of BMP6, known to inhibit proliferation of myeloma cell lines and survival of primary myeloma plasma cells and to confer better prognosis (227);

16. Lower expression of LIME1, the B-cell receptor and B-cell activator gene (28);

17. Higher expression of the inhibitor of apoptosis gene BIRC3 which is a cellular inhibitor of apoptosis 2, cIAP2, a target and regulator of NF-κB signaling, with lower expression in myeloma cells than normal plasma cells (29-30);

18. Higher expression of FER1L4 a trans membrane gene located to chromosome 20q11.23, whose function is as yet unknown; and

19. Higher expression of PLAUR(CD87), which was associated with better survival, in contrast with previous reports (8,31).

The strength of the association between the expression of these genes and post-relapse survival is evident by the ability of 18 of the genes (PLAUR the exception) to predict post relapse survival with a 4.4 hazard ratio. Furthermore, limiting the predictive model to those genes that have a false discovery rate of 5% or better, predicted post relapse survival of the 127 total therapy 2 patients with a hazard ratio of 8.4, of the 32 total therapy 3 patients with a hazard ratio of 5.6, and of the other 98 patients with a hazard ratio of 3.9.

While the association of higher expression of BMP6, CD31, or of the AP-1 complex and lower expression of CCNE2 makes mechanistic sense and higher expression of SIX5 could be signaling MMPC to differentiate, expression levels of other genes are unexpected. A higher expression of HMOX1 and of the inhibitor of apoptosis BIRC3 is associated with drug resistance and shorter survival, however, as demonstrated herein, higher expression of HMOX1 and BIRC3 correlated to an increase in survival of myeloma cells after treatment. In addition, SOCS3 and CISH are both suppressors of cytokine signaling and demonstrate opposite changes in expression levels. Furthermore, TSC22D3, an AP-1 suppressor gene, demonstrated higher expression.

Genes identified as increasing survival of cancer cells post treatment are potential therapeutic targets. Agents, such as chemotherapeutic agents, drugs or other compounds or biomolecules, effective to inhibit or prevent the increase or decrease of expression of the genes that confers post treatment survival to the cancer cells would improve therapeutic efficacy of a treatment regimen, decrease relapse and improve the cancer patient's chance for survival. Potential agents may be known in the art, may be synthesized or may be produced via standard molecular biological techniques. These agents may be tested in assays measuring gene expression levels and/or measuring gene products in cancer cell lines in vitro or in ex vivo samples in the presence or absence of chemotherapeutic agents utilized in known treatment regimens.

While the examples provided herein utilize multiple myeloma cells, one of ordinary skill in the art can see that the methods, systems and kits provided herein are readily adapted to any post-relapse situation. Global gene expression profiling and the statistical analysis techniques provided herein are well-suited to identify genes that are associated with the survival of cancer cells post treatment. The predictive model described herein can be configured for any cancer.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example 1 Methods and Materials Study Subjects

Gene expression profiles (GEP) of CD-138 selected myeloma cells were available on 127 patients with myeloma treated on total therapy 2 protocol (TT2) (32-22) at the time of first relapse (RL); for 71 of these patients, gene expression profiles was also analyzed prior to initiation of therapy (baseline, BL). These gene expression profiles data were used for post relapse survival analysis. Relapsed patients were treated with salvage therapy including thalidomide alone or in combination, lenalidomide alone or in combination, Bortezomib alone or in combination, BTD or BLD with or without chemotherapy (e.g. PACE), DT-PACE or VDT-PACE, or further transplant, as previously reported (34). Plasma cell purifications and gene expression profiles using the Affymetrix U133Plus2.0 microarray (Santa Clara, Calif.), were performed as previously described (35).

Cells for Co-Culture Experiments

Multiple myeloma plasma cells (MMPC) were purified from heparinized bone marrow aspirates obtained from previously untreated patients with active MM during clinic visits, prior to initiation of treatment protocols. Multiple myeloma plasma cells were isolated using CD138 immunomagnetic bead selection and the automated autoMACs Separator (Miltenyi-Biotec, Auburn, Calif.). Multiple myeloma plasma cells purity was determined by CD38/CD45 flow cytometry to be routinely >95%.

Osteoclasts (OC) were prepared as previously described. Briefly, peripheral blood mononuclear cells (PBMC) were obtained from eight MM patients. Signed IRB-approved informed consent forms are kept on record. The cells were cultured at 2.5×10⁶ cells/ml in α-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum, antibiotics, RANKL (50 ng/ml), macrophage colony stimulating factor (M-CSF) (25 ng/ml), and 10 nM dexamethasone (Sigma, St. Louis, Mo.) (osteoclast media) for 10-14 days, at which time they contained large numbers of multinucleated, TRAP positive osteoclasts with bone-resorbing activity (36). RANKL and M-CSF were purchased from PeproTech, Princeton, N.J.

Mesenchymal cells from seven healthy donors were obtained from Darwin Prockop (Texas A & M Health Science Center College of Medicine Institute for Regenerative Medicine at Scott & White in Temple, Tex.). MSC were cultivated according to Dr. Prockop's established laboratory protocols (34).

MMPC and OC Co-Cultures

Osteoclast cultures were washed 3 times with phosphate-buffered saline to detach and remove any remaining non-adherent cells. For testing the molecular consequences of multiple myeloma plasma cells interaction with OC (MMPC/OC), 1.5×10⁶ CD138 sorted multiple myeloma plasma cells in 3 ml of osteoclast medium lacking dexamethasone were added per 30-mm diameter culture plates and the plates incubated for 4 days at 37° C. in a humidified atmosphere containing 5% CO₂. As reported previously, multiple myeloma plasma cells did not adhere to the osteoclasts and were easily recovered from co-cultures by gentle pipetting (33). The purity of recovered myeloma cells was evaluated by flow cytometry using PE-conjugated anti-CD38 and FITC-conjugated anti-CD45 monoclonal antibodies and was routinely ≧95% (FIG. 1). RNA was extracted using RNeasy kit (Qiagen) and DNA digested using RNase free DNase set (Qiagen) (35). RNA was similarly extracted from osteoclasts after co-culture, from OC cultured without multiple myeloma plasma cells, and from 0.5×10⁶ multiple myeloma plasma cells immediately after sorting and used as controls. Eight experiments were performed using multiple myeloma plasma cells from 8 patients and osteoclasts from 8 different patients.

MMPC and MSC Co-Cultures

MSC were seeded in 24-well plates at 40,000 cells per well in complete culture medium at least 24 hours before adding multiple myeloma plasma cells, at which time the medium was removed and 1×10⁶ CD138-sorted (>95% viability as determined by trypan blue exclusion) multiple myeloma plasma cells in complete culture media were added to each well (MMPC/MSC). The plates were kept in a humidified atmosphere at 37° C. and 5% CO₂. After 18 hours incubation, the medium was carefully removed, total RNA extracted using RNeasy kit (Qiagen), and DNA digested using RNase free DNase set (Qiagen). For the control group, the medium was removed from MSC, and 1×10⁶ CD138-sorted (>95% viability) multiple myeloma plasma cells were added per well in phosphate-buffered saline in a total volume of ≦20 μL. Immediately afterward, the MSC+ multiple myeloma plasma cells mixture was lysed, and total RNA was extracted as described above.

Example 2 Analysis Analysis of Global Gene Expression

Global gene expression of multiple myeloma plasma cells/OC and MM/MSC interactions was analyzed using Affymetrix U133Plus2 chips. GeneChip Operating Software normalized output data (CHP files) were further analyzed using Acuity 4 bioinformatics software for analysis of microarrays (Molecular Devices, Sunnyvale, Calif.). To determine changes in gene expression, genes were selected that comply with the following three criteria: paired t-test p-value≦0.05, 500 mean signal cutoff in either pre- or post-co-culture, and at least a two-fold difference in mean signal as calculated by dividing the signal mean following co-culture by the signal mean before co-culture. Thereafter, the datasets selected for MMPC/MSC and MMPC/OC co-cultures were compared in order to identify genes whose expression was similarly changed in both co-culture systems. Ingenuity Pathways Analysis (IPA) software (Ingenuity Systems, Redwood City, Calif.) was used to identify networks of interrelated genes. IPA gene network score is the negative log of right-tailed Fisher's Exact Test p-value.

Survival Analysis

To determine which, if any of the 58 genes, whose expression was changed in co-culture, were related to the clinical course of the disease, GEP of patients in relapse was analyzed using BRB-ArrayTools software (commercially available or available at www.linus.nci.nih.gov/BRB-ArrayTools.html) to identify genes associated with survival of these patients following relapse. A statistical significance level was computed for each gene, dichotomized at the median to low and high signal, based on univariate proportional hazards models (37). These p values were then used in a multivariate permutation test (38-39) in which the survival times and censoring indicators were randomly permutated among arrays. The multivariate permutation test was used to provide 90% confidence that the false discovery rate was less than 10%. The false discovery rate is the proportion of the list of genes claimed to be differentially expressed that are false positives.

To determine whether the extent of change of gene expression was related to outcome, among the 58 genes those were identified whose expression was also changed at relapse compared with baseline and calculated the ratio of change (signal at relapse/baseline signal were associated with post relapse survival. Survival graphs were generated using Kaplan-Meier methods, and the log-rank test was used for comparisons.

Analyses to Evaluate Possible Contamination of MMPC Cells after Coculture by OC

The purity of myeloma cells recovered from co-culture with osteoclasts (>95%) was the same as their purity at the start of the experiments, suggesting that the gene expression observed after co-culture are myeloma genes. Nevertheless, to ascertain that GEP of multiple myeloma plasma cells after co-culture does not represent contamination by osteoclasts or their progenitors, several analyses were performed.

In order to determine if genes expressed by myeloma cells after co-culture could reflect a small contamination of osteoclasts or their progenitors, probe sets were selected that were not expressed by myeloma cells prior to co-culture (detection p-value>0.05 and signal<500 in all 8 samples) and were highly expressed by osteoclasts after co-culture (detection p≦0.05 and signal range 3000-32587 in all 8 OC samples). 42 such probe sets were identified and for each the ratio of signals in multiple myeloma plasma cells after co-culture (signal range 98-32203) to the signals of OC from the same co-cultures was calculated. These ratios varied widely for each co-culture and between co-cultures, from a low of 0.03 to 2.94; there was no correlation between these ratios and osteoclast signal intensity. The median ratio for the 42 probesets across the 8 experiments was 0.4, range 0.1-1.0.

16 probesets were further selected that were expressed by OC after co-culture (mean signal 3008-7990) and multiple myeloma plasma cells prior to co-culture (mean signal 6366-28867). Expression of these probesets by multiple myeloma plasma cells after co-culture was reduced by 50 to 92%. There was no correlation between signal intensities by OC and reduction of expression (r=−0.10027), nor between the levels of expression by multiple myeloma plasma cells before co-culture and the Pre/Post ratios (r=−0.38973). These data clearly indicate that myeloma PC gene expression after co-culture with osteoclasts does not represent a small contamination by osteoclasts.

Example 3

Multiple Myeloma Plasma Cells in Co-Culture with Osteoclasts

Changes in Gene Expression by MMPC Following MMPC/OC Interaction.

Thirteen experiments using primary multiple myeloma plasma cells from eight patients and MSC from five healthy donors were carried out. Survival of multiple myeloma plasma cells in co-culture after 4-7 days was significantly higher (23% average) than controls (p<0.0002, 2-tailed Wilcoxon paired signed-rank test). Expression by myeloma cells of 887 Affymetrix probesets, representing 675 genes, was changed following interaction with osteoclasts (552 genes up regulated and 123 down regulated). Ingenuity Pathways Analysis software assigned 605 of these genes to 40 networks of interrelated genes, of them 33 with high IPA score in the range 8-42.

Differentially Expressed Genes in MMPC/MSC Interaction

Following interaction of multiple myeloma plasma cells with MSC, expression of 365

Affymetrix probesets, corresponding to 296 genes (161 up regulated and 135 down regulated) was changed (Table 1). Ingenuity Pathways Analysis software assigned 244 of these 296 genes to 19 networks of interrelated genes, of them 16 with high IPA score in the range 12-41.

TABLE 1 Genes whose expression in myleoma cells changed after co-cultue with mesenchymal stem cells (MSC) Affymetrix id Gene Symbol Chromosomal Location 228038_at SOX2 chr3q26.3-q27 211506_s_at IL8 chr4q13-q21 204614_at SERPINB2 chr18q21.3 206336_at CXCL6 chr4q21 206337_at CCR7 chr17q12-q21.2 209277_at TFPI2 chr7q22 235638_at RASSF6 chr4q13.3 202859_x_at IL8 chr4q13-q21 219971_at IL21R chr16p11 221658_s_at IL21R chr16p11 223333_s_at ANGPTL4 chr19p13.3 209278_s_at TFPI2 chr7q22 223704_s_at DMRT2 chr9p24.3 1554997_a_at PTGS2 chr1q25.2-q25.3 221009_s_at ANGPTL4 chr19p13.3 209732_at CLEC2B chr12p13-p12 39402_at IL1B chr2q14 205266_at LIF chr22q12.2 202510_s_at TNFAIP2 chr14q32 213865_at DCBLD2 chr3q12.1|3 211003_x_at TGM2 chr20q12 213506_at F2RL1 chr5q13 204748_at PTGS2 chr1q25.2-q25.3 227314_at ITGA2 chr5q23-q31 202638_s_at ICAM1 chr19p13.3-p13.2 238513_at PRRG4 chr11p13 210143_at ANXA10 chr4q33 203665_at HMOX1 chr22q12|22q13.1 217165_x_at MT1F chr16q13 227697_at SOCS3 chr17q25.3 208581_x_at MT1X chr16q13 211573_x_at TGM2 chr20q12 202637_s_at ICAM1 chr19p13.3-p13.2 222068_s_at LRRC50 chr16q24.1 215485_s_at ICAM1 chr19p13.3-p13.2 213338_at TMEM158 chr3p21.3 215034_s_at TM4SF1 chr3q21-q25 204595_s_at STC1 chr8p21-p11.2 244721_at TP53INP1 chr8q22 206298_at ARHGAP22 chr10q11.22 205067_at IL1B chr2q14 201578_at PODXL chr7q32-q33 206432_at HAS2 chr8q24.12 206574_s_at LOC100131062 /// chr8q24.3 PTP4A3 211668_s_at PLAU chr10q24 204338_s_at RGS4 chr1q23.3 205968_at KCNS3 chr2p24 204011_at SPRY2 chr13q31.1 204115_at GNG11 chr7q21 223986_x_at DMRT2 chr9p24.3 204597_x_at STC1 chr8p21-p11.2 221239_s_at FCRL2 chr1q21 208025_s_at HMGA2 chr12q15 201860_s_at PLAT chr8p12 230372_at HAS2 chr8q24.12 202888_s_at ANPEP chr15q25-q26 206461_x_at MT1H chr16q13 216336_x_at MT1E /// MT1H /// chr16q13 /// chr1q43 MT1M /// MT1P2 219682_s_at TBX3 chr12q24.1 208937_s_at ID1 chr20q11 218451_at CDCP1 chr3p21.31 209621_s_at PDLIM3 chr4q35 205100_at GFPT2 chr5q34-q35 201650_at JUP /// KRT19 chr17q21 /// chr17q21.2 212185_x_at MT2A chr16q13 204745_x_at MT1G chr16q13 201625_s_at INSIG1 chr7q36 1560477_a_at SAMD11 chr1p36.33 210689_at CLDN14 chr21q22.3 211502_s_at PFTK1 chr7q21-q22 209387_s_at TM4SF1 chr3q21-q25 211924_s_at PLAUR chr19q13 218000_s_at PHLDA1 chr12q15 223222_at SLC25A19 chr17q25.3 213638_at PHACTR1 chr6p24.1 210538_s_at BIRC3 chr11q22 209695_at LOC100131062 /// chr8q24.3 PTP4A3 209803_s_at PHLDA2 chr11p15.5 210916_s_at CD44 chr11p13 212859_x_at MT1E chr16q13 220994_s_at STXBP6 chr14q12 205032_at ITGA2 chr5q23-q31 201042_at TGM2 chr20q12 235548_at APCDD1L chr20q13.32 226462_at STXBP6 chr14q12 205924_at RAB3B chr1p32-p31 214430_at GLA chrXq22 201169_s_at BHLHE40 chr3p26 204339_s_at RGS4 chr1q23.3 219168_s_at PRR5 chr22q13 36711_at MAFF chr22q13.1 219134_at ELTD1 chr1p33-p32 221581_s_at LAT2 chr7q11.23 213256_at MARCH3 chr5q23.2 203180_at ALDH1A3 chr15q26.3 237411_at ADAMTS6 chr5q12 201920_at SLC20A1 chr2q11-q14 217279_x_at MMP14 chr14q11-q12 206953_s_at LPHN2 chr1p31.1 223101_s_at ARPC5L chr9q33.3 203068_at KLHL21 chr1p36.31 210845_s_at PLAUR chr19q13 226064_s_at DGAT2 chr11q13.5 211456_x_at MT1P2 chr1q43 223537_s_at WNT5B chr12p13.3 216250_s_at LPXN chr11q12.1 201976_s_at MYO10 chr5p15.1-p14.3 205207_at IL6 chr7p21 232122_s_at VEPH1 chr3q24-q25 224009_x_at DHRS9 chr2q31.1 212014_x_at CD44 chr11p13 221664_s_at F11R chr1q21.2-q21.3 203821_at HBEGF chr5q23 205034_at CCNE2 chr8q22.1 209386_at TM4SF1 chr3q21-q25 224911_s_at DCBLD2 chr3q12.1|3 204490_s_at CD44 chr11p13 217127_at CTH chr1p31.1 228082_at ASAM chr11q24.1 210896_s_at ASPH chr8q12.1 226059_at TOMM40L chr1q23.3 212746_s_at CEP170 chr1q44 202748_at GBP2 chr1p22.2 1557905_s_at CD44 chr11p13 209835_x_at CD44 chr11p13 1555673_at KAP2.1B /// chr17q12-q21 /// KRTAP2-4 /// chr17q21.2 LOC644350 /// LOC728285 /// LOC728934 /// LOC730755 203811_s_at DNAJB4 chr1p31.1 227340_s_at RGMB chr5q21.1 219634_at CHST11 chr12q 232861_at PDP2 chr16q22.1 204445_s_at ALOX5 chr10q11.2 228703_at P4HA3 chr11q13.4 218368_s_at TNFRSF12A chr16p13.3 1554097_a_at LOC554202 chr9p21.3 201037_at PFKP chr10p15.3-p15.2 227123_at RAB3B chr1p32-p31 230799_at LOC100134259 chr2p21 1552717_s_at CEP170 /// CEP170L chr1q44 /// chr4q26 219257_s_at SPHK1 chr17q25.2 223019_at FAM129B chr9q34.11 204612_at PKIA chr8q21.12 204446_s_at ALOX5 chr10q11.2 209526_s_at HDGFRP3 chr15q25.2 202134_s_at WWTR1 chr3q23-q24 211343_s_at COL13A1 chr10q22 219985_at HS3ST3A1 chr17p12-p11.2 217999_s_at PHLDA1 chr12q15 224097_s_at F11R chr1q21.2-q21.3 206584_at LY96 chr8q21.11 214577_at MAP1B chr5q13 204224_s_at GCH1 chr14q22.1-q22.2 230290_at SCUBE3 chr6p21.3 201629_s_at ACP1 chr2p25 204604_at PFTK1 chr7q21-q22 223618_at FMN2 chr1q43 204749_at NAP1L3 chrXq21.3-q22 223952_x_at DHRS9 chr2q31.1 212003_at C1orf144 chr1p36.13 237563_s_at LOC440731 chr1q42.2 228776_at GJC1 chr17q21.31 213988_s_at SAT1 chrXp22.1 228266_s_at HDGFRP3 chr15q25.2 217998_at LOC652993 /// PHLDA1 chr12q15 /// chr12q21 201739_at SGK1 chr6q23 205479_s_at PLAU chr10q24 228415_at AP1S2 chrXp22.2 209524_at HDGFRP3 chr15q25.2 228253_at LOXL3 chr2p13 203904_x_at CD82 chr11p11.2 227405_s_at FZD8 chr10p11.21 209897_s_at SLIT2 chr4p15.2 205428_s_at CALB2 chr16q22.2 210513_s_at VEGFA chr6p12 219926_at POPDC3 chr6q21 223249_at CLDN12 chr7q21 230792_at FAAH2 chrXp11.1 206571_s_at MAP4K4 chr2q11.2-q12 206085_s_at CTH chr1p31.1 202132_at WWTR1 chr3q23-q24 209032_s_at CADM1 chr11q23.2 203939_at NT5E chr6q14-q21 209676_at TFPI chr2q32 238542_at ULBP2 chr6q25 223961_s_at CISH chr3p21.3 213310_at EIF2C2 chr8q24 212662_at PVR chr19q13.2 224774_s_at NAV1 chr1q32.3 225827_at EIF2C2 chr8q24 47069_at PRR5 chr22q13 223614_at MMP16 chr8q21.3 1555638_a_at SAMSN1 chr21q11 221845_s_at CLPB chr11q13.4 222548_s_at MAP4K4 chr2q11.2-q12 1569003_at TMEM49 chr17q23.1 216693_x_at HDGFRP3 chr15q25.2 206710_s_at EPB41L3 chr18p11.32 220161_s_at EPB41L4B chr9q31-q32 201422_at IFI30 chr19p13.1 208708_x_at EIF5 chr14q32.32 219878_s_at KLF13 chr15q12 1562460_at CNDP2 chr18q22.3 1553995_a_at NT5E chr6q14-q21 236656_s_at LOC100130506 — 204220_at GMFG chr19q13.2 202869_at OAS1 chr12q24.1 210655_s_at FOXO3 /// ZNF286C chr17p11.2 /// chr6q21 221527_s_at PARD3 chr10p11.22-p11.21 206809_s_at HNRNPA3 /// chr10q11.21 /// chr2q31.2 HNRNPA3P1 205469_s_at IRF5 chr7q32 235010_at LOC729013 chr11p15.3 230128_at IGL@ chr22q11.1-q11.2 222621_at DNAJC1 chr10p12.31 226659_at DEF6 chr6p21.33-p21.1 228291_s_at NCRNA00153 chr20pter-q11.23 213521_at PTPN18 chr2q21.1 209282_at PRKD2 chr19q13.3 218747_s_at TAPBPL chr12p13.31 206641_at TNFRSF17 chr16p13.1 204821_at BTN3A3 chr6p21.3 214209_s_at ABCB9 chr12q24 203615_x_at SULT1A1 chr16p12.1 225941_at EIF4E3 chr3p14 225728_at SORBS2 chr4q35.1 202265_at BMI1 chr10p11.23 220998_s_at UNC93B1 chr11q13 213540_at HSD17B8 chr6p21.3 212093_s_at MTUS1 chr8p22 236436_at SLC25A45 chr11q13.1 239412_at IRF5 chr7q32 209291_at ID4 chr6p22-p21 206170_at ADRB2 chr5q31-q32 216981_x_at SPN chr16p11.2 230388_s_at LOC644246 chr17q21.31 227087_at INPP4A chr2q11.2 209357_at CITED2 chr6q23.3 222762_x_at LIMD1 chr3p21.3 203837_at MAP3K5 chr6q22.33 219441_s_at LRRK1 chr15q26.3 221989_at RPL10 chrXq28 226104_at RNF170 chr8p11.21 227817_at PRKCB chr16p11.2 225981_at C17orf28 chr17q25.1 226452_at PDK1 chr2q31.1 242414_at QPRT chr16p11.2 235475_at LOC100129720 chr3q25.1 219541_at LIME1 chr20q13.3 225136_at PLEKHA2 chr8p11.23 201041_s_at DUSP1 chr5q34 205504_at BTK chrXq21.33-q22 215299_x_at SULT1A1 chr16p12.1 209230_s_at NUPR1 chr16p11.2 203317_at PSD4 chr2q13 204118_at CD48 chr1q21.3-q22 227134_at SYTL1 chr1p36.11 229009_at SIX5 chr19q13.32 202075_s_at PLTP chr20q12-q13.1 225763_at RCSD1 chr1q22-q24 203408_s_at SATB1 chr3p23 207777_s_at SP140 chr2q37.1 219865_at HSPC157 /// chr1p36.12 LOC100128919 204552_at INPP4A chr2q11.2 207957_s_at PRKCB chr16p11.2 218058_at CXXC1 chr18q12 227429_at EFCAB4A chr11p15.5 217192_s_at PRDM1 chr6q21-q22.1 231647_s_at FCRL5 chr1q21 208983_s_at PECAM1 chr17q23 220565_at CCR10 chr17q21.1-q21.3 206589_at GFI1 chr1p22 225721_at SYNPO2 chr4q26 226489_at TMCC3 chr12q22 226132_s_at MANEAL chr1p34.3 223569_at PPAPDC1B chr8p12 33304_at ISG20 chr15q26 203153_at IFIT1 chr10q25-q26 219143_s_at RPP25 chr15q24.1 212096_s_at MTUS1 chr8p22 232213_at PELI1 chr2p13.3 206176_at BMP6 chr6p24-p23 1559584_a_at C16orf54 /// chr16p11.2 hCG_1644884 218437_s_at LZTFL1 chr3p21.3 223358_s_at PDE7A chr8q13 228897_at DERL3 chr22q11.23 229390_at FAM26F chr6q22.1 221727_at SUB1 chr5p13.3 219961_s_at NCRNA00153 chr20pter-q11.23 229497_at ANKDD1A chr15q22.31 209827_s_at IL16 chr15q26.3 218494_s_at SLC2A4RG chr20q13.33 208981_at PECAM1 chr17q23 215923_s_at PSD4 chr2q13 213005_s_at KANK1 chr9p24.3 236226_at BTLA chr3q13.2 219014_at PLAC8 chr4q21.22 226140_s_at OTUD1 chr10p12.2 227074_at LOC100131564 chr1p22.1 224724_at SULF2 chr20q12-q13.2 208982_at PECAM1 chr17q23 229530_at GUCY1A3 chr4q31.3-q33|4q31.1-q31.2 208056_s_at CBFA2T3 chr16q24 223044_at SLC40A1 chr2q32 205671_s_at HLA-DOB chr6p21.3 206150_at CD27 chr12p13 224722_at MIB1 chr18q11.2 204698_at ISG20 chr15q26 207001_x_at TSC22D3 chrXq22.3 203111_s_at PTK2B chr8p21.1 219371_s_at KLF2 chr19p13.13-p13.11 226384_at PPAPDC1B chr8p12 218048_at COMMD3 chr10pter-q22.1 222245_s_at FER1L4 chr20q11.22 201465_s_at JUN chr1p32-p31 203836_s_at MAP3K5 chr6q22.33 227865_at C9orf103 chr9q21-q22 226646_at KLF2 chr19p13.13-p13.11 203110_at PTK2B chr8p21.1 212225_at EIF1 chr17q21.2 235401_s_at FCRLA chr1q23.3 218409_s_at DNAJC1 chr10p12.31 227711_at GTSF1 chr12q13.2 207794_at CCR2 /// FLJ78302 chr3p21.31 201694_s_at EGR1 chr5q31.1 51158_at FAM174B chr15q26.1 225579_at PQLC3 chr2p25.1 225895_at SYNPO2 chr4q26 226558_at LOC653071 — 219525_at SLC47A1 chr17p11.2 210889_s_at FCGR2B chr1q23 213160_at DOCK2 chr5q35.1 227404_s_at EGR1 chr5q31.1 227641_at FBXL16 chr16p13.3 205804_s_at TRAF3IP3 chr1q32.3-q41 206478_at KIAA0125 chr14q32.33 207245_at UGT2B17 chr4q13 226344_at ZMAT1 chrXq21 221666_s_at PYCARD chr16p12-p11.2 205901_at PNOC chr8p21 207980_s_at CITED2 chr6q23.3 207321_s_at ABCB9 chr12q24 201466_s_at JUN chr1p32-p31 233555_s_at SULF2 chr20q12-q13.2 204794_at DUSP2 chr2q11 205774_at F12 chr5q33-qter 1554208_at MEI1 chr22q13.2 204192_at CD37 chr19q13.3 227235_at GUCY1A3 chr4q31.3-q33|4q31.1-q31.2 213888_s_at LOC100133233 /// chr1q32.2 /// chr1q32.3-q41 TRAF3IP3 1568964_x_at SPN chr16p11.2 221297_at GPRC5D chr12p13.3 225792_at HOOK1 chr1p32.1 206687_s_at PTPN6 chr12p13 207996_s_at C18orf1 chr18p11.2 225720_at SYNPO2 chr4q26 206978_at CCR2 /// FLJ78302 chr3p21.31 218918_at MAN1C1 chr1p35 232304_at PELI1 chr2p13.3 201044_x_at DUSP1 chr5q34 211998_at H3F3A /// H3F3B /// chr17q25 /// chr1q41 /// LOC440926 chr2q31.1 226884_at LRRN1 chr3p26.2 206121_at AMPD1 chr1p13 230011_at MEI1 chr22q13.2 202340_x_at NR4A1 chr12q13 220377_at FAM30A chr14q32.33 209189_at FOS chr14q24.3 202768_at FOSB chr19q13.32

Differentially Expressed Genes by MMPC Common to Both Co-Culture Systems:

Comparison of genes whose expression was changed in multiple myeloma plasma cells following co-culture with osteoclasts and genes whose expression was similarly changed in MMPC/MSC co-culture identified 72 commonly changed probesets, representing 58 genes; 33 genes were up regulated and 25 down regulated. The 58 genes include one cytokine, 12 transcription regulators, two growth factors, 16 enzymes, five receptors, one transporter and 22 with other functions (Table 2). Using IPA, 54 of the 58 genes (72 probesets) were assigned to five distinguished networks on interrelated genes with high probability IPA scores (FIGS. 2A-2E).

TABLE 2 72 probe sets whose expression was similarly altered in MMPC following co-culture with osteoclasts and osteoblasts Ratio of means Ratio of means MM/OC MM/MSC after/before after/before Type Affymetrix ID Symbol interaction interaction Cytokine 202859_x_at IL8 50 5.56 211506_s_at IL8 50 11.11 Phosphatase 201044_x_at DUSP1 0.26 0.24 201041_s_at DUSP1 0.08 0.48 Peptidases 202888_s_at ANPEP 14.29 2.7 217279_x_at MMP14 4 2.27 205479_s_at PLAU 7.69 1.92 211668_s_at PLAU 9.09 2.94 Other enzymes 206121_at AMPD1 0.21 0.23 207245_at UGT2B17 0.21 0.33 204698_at ISG20 0.43 0.39 33304_at ISG20 0.44 0.43 201422_at IFI30 5.88 1.82 213988_s_at SAT1 3.03 1.96 219634_at CHST11 2.04 2.08 214430_at GLA 2.86 2.38 201042_at TGM2 20 2.44 210538_s_at BIRC3 2.5 2.5 203665_at HMOX1 9.09 3.33 G-protein 206978_at CCR2 0.31 0.26 coupled 207794_at CCR2 0.28 0.37 receptors 206337_at CCR7 2.13 6.25 221297_at GPRC5D 0.31 0.28 Growth factors 206176_at BMP6 0.36 0.43 203821_at HBEGF 3.45 2.17 Kinase 201739_at SGK1 7.14 1.96 219257_s_at SPHK1 2.38 2.04 Transcription 202768_at FOSB 0.05 0.03 regulators 209189_at FOS 0.07 0.07 207980_s_at CITED2 0.3 0.33 226646_at KLF2 0.43 0.37 201465_s_at JUN 0.27 0.38 219371_s_at KLF2 0.26 0.39 207001_x_at TSC22D3 0.19 0.39 208056_s_at CBFA2T3 0.47 0.4 213005_s_at KANK1 0.45 0.41 218494_s_at SLC2A4RG 0.45 0.42 229009_at SIX5 0.28 0.47 209357_at CITED2 0.42 0.49 36711_at MAFF 2.56 2.33 Transmembrane 210845_s_at PLAUR 11.11 2.22 receptors 211924_s_at PLAUR 8.33 2.56 215485_s_at ICAM1 3.33 3.13 202637_s_at ICAM1 3.85 3.23 202638_s_at ICAM1 4.55 3.45 1557905_s_at CD44 3.85 2.08 209835_x_at CD44 4.55 2.08 212014_x_at CD44 5 2.17 208982_at PECAM1 0.45 0.4 208981_at PECAM1 0.47 0.41 208983_s_at PECAM1 0.39 0.45 Transporter 223222_at SLC25A19 3.03 2.5 Other 236226_at BTLA 0.48 0.41 205034_at CCNE2 3.23 2.17 204118_at CD48 0.35 0.47 203904_x_at CD82 3.7 1.92 212746_s_at CEP170 4.76 2.13 223961_s_at CISH 2.78 1.85 206710_s_at EPB41L3 33.33 1.82 223019_at FAM129B 6.25 2.04 222245_s_at FER1L4 0.42 0.38 203068_at KLHL21 2.94 2.27 219541_at LIME1 0.25 0.48 226884_at LRRN1 0.31 0.23 212859_x_at MT1E 2.5 2.44 206461_x_at MT1H 2.94 2.7 219014_at PLAC8 0.47 0.41 238513_at PRRG4 2.63 3.33 227697_at SOCS3 3.45 3.23 1569003_at TMEM49 4.17 1.85 202510_s_at TNFAIP2 4.76 3.7 206641_at TNFRSF17 0.43 0.5

To investigate if these 58 genes are relevant to the biology of myeloma as expressed by correlation with the clinical course of the disease, those genes whose expression by myeloma cells obtained at relapse is significantly associated with post relapse survival were identified. Of the 58 genes, 22 genes (27 probesets, Table 3) changed expression after relapse compared with baseline in the 71 relapsed patients treated on TT2 for whom baseline and relapse GEP were available. The change in expression of these 72 probesets was calculated as the ratios of signal at relapse/baseline signal. Ratios of 8 probesets, representing 7 genes, dichotomized at the median, were significantly associated with survival at 0.05 level of univariate analysis. These probesets are listed in Table 4 in order of the univariate test p-value.

TABLE 3 27 probesets whose expression changed following co-culture also changed at relapse* Baseline Relapse p Ratio RL/BL Gene Median Median (Paired Median Probe set Symbol (range) (range) t-Test) (range) 208056_s_at CBFA2T3 1479 1285 <0.001 0.96 (444-4510)  (251-2824)  (0.03-5.4)  207980_s_at CITED2 6341 5268 0.001 0.89 (415-31118) (277-22292) (0.008-1.80)  209357_at CITED2 8893 7636 0.009 0.71  (93-22490)  (12-23638) (0.04-5.87) 201041_s_at DUSP1 24359  22540  0.049 0.83 (1892-42774)  (1075-48866)  (0.26-1.88) 221297_at GPRC5D 7488 6228 0.029 0.63 (709-27040) (504-29988) (0.02-2.15) 213005_s_at KANK1 6885 4470 0.000 0.86 (900-17834) (650-27206) (0.04-2.08) 226646_at KLF2  852  736 0.001 0.91 (193-3233)  (134-2376)  (0.02-6.20) 208981_at PECAM1 9833 6547 0.000 0.80 (323-19790) (1343-15633)  (0.001-3.51)  208982_at PECAM1 9785 8288 0.005 0.73 (535-41487) (285-36351) (0.05-5.33) 208983_s_at PECAM1 4679 3412 0.023 0.88 (174-15539)  (18-11671) (0.10-5.71) 206641_at TNFRSF17 20293  18747  0.037 0.90 (6980-37358)  (166-32405) (0.19-2.49) 203665_at HMOX1  326  780 0.000 1.32 (25-2747)  (83-18250)  (0.18-11.44) 203821_at HBEGF    106.906  158 0.004 1.17 (21-815)  (21-4625)   (0.11-13.93) 206710_s_at EPB41L3     45.167  82 0.010 0.69 (3-414)  (5-1604) (0.10-5.4)  203068_at KLHL21    111.004  193 0.000 1.48 (43-1005) (14-1427)  (0.14-15.54) 201422_at IFI30 1381 1780 0.001 2.22 (102-7766)  (317-18411)  (0.09-88.54) 215485_s_at ICAM1  181  297 0.003 1.62 (28-1172) (28-1384)  (0.17-30.45) 223222_at SLC25A19  465  627 0.000 1.30 (32-1550) (219-1521)  (0.28-7.35) 219634_at CHST11  780 1072 0.001 0.68 (166-2131)  (65-4702) (0.05-8.12) 205479_s_at PLAU  74  97 0.016 1.63 (23-732)  (26-869)   (0.02-21.17) 202638_s_at ICAM1  448  573 0.048 0.79 (113-2607)  (55-4685)  (0.06-14.55) 214430_at GLA  756  833 0.008 0.74 (259-2904)  (401-4876)  (0.05-4.94) 223961_s_at CISH  316  266 0.03 0.89 (19-3165) (10-1127)   (0-1.8) 202859_x_at IL8  319  223 0.047 1.48 (59-1672) (41-1266)  (0.13-15.53) 211506_s_at IL8  83  62 0.03 2.22  (6-684) (5-294)  (0.09-88.54) 206461_x_at MT1H  216  149 0.049 1.63 (32-1013) (27-1660)  (0.01-21.17) 211924_s_at PLAUR  305  179 0.01 1.14 (50-1421)  (9-1179) (0.17-6.33) *Of the 72 probesets whose expression by myeloma plasma cells was altered after co-culture, expression of 27 was also changed at relapse compared with baseline.

TABLE 4 Genes whose change in expression correlated with post-relapse survival. Gene Parametric Permutation Hazard Signal Ratio^(&) Probe set symbol p-value p-value* Ratio^(#) median (range) 205034_at CCNE2 0.017 0.0163 1.6 1.04 (0.27-7.69) 208983_s_at PECAM1 0.031 0.0307 0.45 0.92 (0.016-6.2) 203068_at KLHL21 0.032 0.0342 1.321 1.54 (0.06-15.54) 202638_s_at ICAM1 0.038 0.0428 1.546 1.2 (0.11-13.9) 211668_s_at PLAU 0.039 0.0384 1.377 1 (0.05-13.67) 202888_s_at ANPEP 0.042 0.044 0.591 0.92 (0.49-10.23) 205479_s_at PLAU 0.043 0.045 0.631 1.2 (0.28-7.35) 201044_x_at DUSP1 0.049 0.051 0.68 1.22 (0.007-15.73) *Permutation p-values for significant genes were computed based on 10000 random permutations. ^(#)Hazard ratio is the ratio of hazards for a two-fold change in the gene expression level. ^(&)Ratio was calculated as signal at relapse/baseline signal.

Since expression ratios do not reflect signal intensities, it also was determined whether expression signals of the 72 probe sets at relapse, each probe set dichotomized at the median, was associated with post relapse survival of the 127 TT2 patients. BRB ArrayTools identified 21 probesets (18 genes), significantly associated with survival, with a univariate p value of <0.05; the probe sets are listed in Table 5. BRB ArrayTools also used 20 of these probe sets (17 genes) to predict post relapse survival, with a hazard ratio of 4.4 (FIGS. 3A-3C).

TABLE 5 Genes whose expession is significantly associated with survival Gene Parametric Permutation * Hazard Signal Probe set symbol p-value p-value Ratio^(#) Median (range) 205034_at CCNE2    <1e−07 <1e−07 1.828 575 (167-4339) 208982_at PECAM1  2e−7 <1e−07 0.635 9126 (285-36351) 208983_s_at PECAM1 2.3e−6 <1e−07 0.77 3638 (18-15865) 202768_at FOSB 4.8e−6 <1e−07 0.757 1757 (29-12913) 208981_at PECAM1 5.1e−5 <1e−07 0.738 6947 (134-15633) 203665_at HMOX1 8.2e−5 <1e−07 0.721 869 (83-22175) 223961_s_at CISH^($) 1.7e−4 1e−4 0.757 237 (11-2705) 201465_s_at JUN 8.2e−4 9e−4 0.782 1354 (46-11121) 229009_at SIX5 0.001277 0.001  0.745 614 (26-2971) 201044_x_at DUSP1 0.0023 0.0022 0.815 1122 (10-7771) 203821_at HBEGF 0.0046 0.0049 1.219 158 (21-4625) 206176_at BMP6 0.0065 0.0069 0.735 4726 (405-21385) 219541_at LIME1 0.0075 0.007  1.436 1287 (80-4241) 210538_s_at BIRC3 0.011 0.0118 0.88 2760 (21-23688) 201041_s_at DUSP1 0.015 0.0143 0.792 22829 (1075-50162) 222245_s_at FER1L4 0.016 0.0173 0.712 1377 (262-8790) 227697_at SOCS3 0.020 0.0216 1.108 111 (3-3334) 207001_x_at TSC22D3 0.040 0.0396 0.839 1344 (81-9652) 36711_at MAFF 0.042 0.0494 1.189 405 (8-9428) 210845_s_at PLAUR^($) 0.047 0.0468 0.782 430 (45-1548) 203068_at KLHL21 0.048 0.0497 1.18 214 (4-2134) * Permutation p-values for significant genes were computed based on 10000 random permutations. ^(#)Hazard ratio is the ratio of hazards for a two-fold change in the gene expression level. ^($)Expression of these genes at relapse was lower than baseline, whereas their expression was higher after co-culture.

This set of genes from the 13 probeset model was further refined to the 11 genes and their chromosome locations shown in Table 6. Expression of 11 genes with a false discovery rate of ≦5% predicted post relapse survival with a hazard ratio of 8.4 and p<0.0001 of the 127 patients (FIG. 3A). Moreover, of the 127 TT2 patients, this model predicted post relapse survival of 32 relapsed patients following TT3 treatment (FIG. 3B), and of 98 patients who relapsed after achieving complete or near complete remission on several treatment regimens (FIG. 3C), 91 of them including high-dose therapy, suggesting that expression of the selected genes is universally associated with survival following relapse.

TABLE 6 11 genes (13 probesets) whose expression at first relapse predicts post-relapse survival. Probeset Gene Symbol Chromosome location 203665_at HMOX1 chr22q12|22q13.1 203821_at HBEGF chr5q23 205034_at CCNE2 chr8q22.1 223961_s_at CISH chr3p21.3 201044_x_at DUSP1 chr5q34 201465_s_at JUN chr1p32-p31 202768_at FOSB chr19q13.32 206176_at BMP6 chr6p24-p23 208981_at PECAM1 chr17q23 208982_at PECAM1 chr17q23 208983_s_at PECAM1 chr17q23 219541_at LIME1 chr20q13.3 229009_at SIX5 chr19q13.32 A set of 33 probe sets, identified by coculture with osteoclasts, was found to predict post relapse survival. The 33 probe sets are given in Table 7

TABLE 7 33 Probe sets predictive of post-relapse survival Chromosomal No Gene Id Symbol Weights (w_(i))* p-value Location 1 219073_s_at OSBPL10 −0.093019   <1e−07 chr3p22.3 2 205034_at CCNE2 0.039816 1.00E−07 chr8q22.1 3 211764_s_at UBE2D1 0.019541 2.00E−07 chr10q11.2-q21 4 208982_at PECAM1 −0.057215 1.50E−06 chr17q23 5 203382_s_at APOE −0.019823 7.10E−06 chr19q13.2 6 201443_s_at ATP6AP2 0.015442 1.44E−05 chrXp11.4 7 204944_at PTPRG 0.021603 1.82E−05 chr3p21-p14 8 219540_at ZNF267 0.018927 1.86E−05 chr16p11.2 9 208983_s_at PECAM1 −0.088241 2.84E−05 chr17q23 10 203665_at HMOX1 −0.003104 3.11E−05 chr22q12|22q13.1 11 216841_s_at SOD2 0.022577 3.72E−05 chr6q25.3 12 201266_at TXNRD1 0.011841 3.79E−05 chr12q23-q24.1 13 216942_s_at CD58 0.032742 4.72E−05 chr1p13 14 223344_s_at MS4A7 −0.002062 7.40E−05 chr11q12 15 214146_s_at PPBP −0.036276 9.09E−05 chr4q12-q13 16 200878_at EPAS1 −0.018851 1.39E−04 chr2p21-p16 17 213746_s_at FLNA 0.052525 1.48E−04 chrXq28 18 210753_s_at EPHB1 −0.052594 1.84E−04 chr3q21-q23 19 206214_at PLA2G7 −0.003955 2.13E−04 chr6p21.2-p12 20 223961_s_at CISH −0.066213 2.38E−04 chr3p21.3 21 213112_s_at SQSTM1 −0.012277 2.66E−04 chr5q35 22 228377_at KLHL14 −0.097634 2.90E−04 chr18q12.1 23 202872_at ATP6V1C1 0.014609 2.95E−04 chr8q22.3 24 217047_s_at FAM13A −0.063961 3.12E−04 chr4q22.1 25 208981_at PECAM1 −0.059574 3.53E−04 chr17q23 26 224901_at SCD5 −0.072739 3.72E−04 chr4q21.22 27 212737_at GM2A −0.018788 4.11E−04 chr5q31.3-q33.1 28 228759_at CREB3L2 −0.045327 4.36E−04 chr7q34 29 203645_s_at CD163 −0.009919 4.82E−04 chr12p13.3 30 235706_at CPM −0.005803 5.01E−04 chr12q14.3 31 205057_s_at IDUA −0.050522 5.09E−04 chr4p16.3 32 219888_at SPAG4 −0.041312 5.64E−04 chr20q11.21 33 210785_s_at C1orf38 −0.027797 5.85E−04 chr1p35.3 Post relapse survival rates for high and low risk patients from different groups evaluated by GEP and the 33 gene model in Table 7 are shown in FIGS. 5A-5C. A sample was predicted as high risk if its prognostic index was >0.794828, computed by the formula: PI=Σ_(i)w_(i)x_(i)+6.999731, where w_(i) and x_(i) are the weight and logged gene expression for the i^(th) gene.

The following references are cited herein.

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While the invention has been described with reference to certain particular embodiments, those skilled in the art will appreciate that various modifications may be made without departing from the spirit and scope of the invention.

All patents and publications mentioned in this specification are indicative of the level of those skilled in the art to which the invention pertains. All patents, publications, and sequence references (such as gene names and reference acesison numbers, including their underlying human gene sequences and associated annotations in databases such as the NCBI Entrez portal) herein are incorporated by reference to the same extent as if each individual reference was specifically and individually indicated as having been incorporated by reference in its entirety. Where there is any conflict between a reference incorporated by reference and this application, this application will control.

Headings used in this application are for convenience only and do not affect the interpretation of this application. 

1. A method for predicting post-relapse survival of a relapsed multiple myeloma patient, comprising testing the level of gene expression of a group of multiple myeloma genes in multiple myeloma plasma cells obtained from the patient, thereby predicting the post-relapse survival of the patient, wherein the group of multiple myeloma genes comprises at least CCNE2, PECAM1, HMOX1, and CISH; optionally wherein the group of multiple myeloma genes further comprises HBEGF, JUN, SIX5, and DUSP1; optionally wherein the group of multiple myeloma genes further comprises BMP6, FOSB, and LIME1.
 2. The method of claim 1, wherein: a decrease in expression of PECAM1, HMOX1, CISH, SIX5, BMP6, JUN, FOSB, and/or DUSP1; and/or an increase in expression of LIME1, CCNE2, and/or HBEGF, is predictive of a low survival rate of the patient.
 3. The method of claim 1, wherein the group of multiple myeloma genes further comprises BIRC3, FER1L4, TSC22D3, MAFF, SOCS3, and KLHL21.
 4. The method of claim 3, wherein: a decrease in the expression level of BIRC3, FER1L4, and/or TSC22D3; and/or an increase in expression level of MAFF, SOCS3 and/or KLHL21, is predictive of a low survival rate of the patient.
 5. A method for predicting post-relapse survival of a relapsed multiple myeloma patient, comprising testing the level of gene expression of a group of multiple myeloma genes in plasma cells obtained from the patient, thereby predicting the post-relapse survival of the patient, wherein the multiple myeloma genes comprises all of OSBPL10, CCNE2, UBE2D1, PECAM1, APOE, ATP6AP2, PTPRG, ZNF267, HMOX1, SOD2, TXNRD1, CD58, MS4A7, PPBP, EPAS1, FLNA, EPHB1, PLA2G7, CISH, SQSTM1, KLHL14, ATP6V1C1, FAM13A, SCD5, GM2A, CREB3L2, CD163, CPM, IDUA, SPAG4, and Clorf38.
 6. The method of claim 5, wherein the method comprises determining a disease index that is a weighted sum the levels of gene expression for the multiple myeloma genes.
 7. The method of claim 6, wherein the levels of gene expression are log transformed.
 8. The method of claim 7, wherein the weights for the logged gene expression levels are about the weights given in Table
 7. 9. The method of claim 8, wherein a disease index above about 0.79 indicates that the subject has a low survival rate, where the disease index is calculated by the formula: DI=Σ_(i)w_(i)x_(i)+6.999731, where w_(i) and x_(i) are the weight and logged gene expression for the i^(th) gene.
 10. A method for predicting post-relapse survival of a relapsed multiple myeloma patient, comprising testing the level of gene expression of a group of multiple myeloma genes in plasma cells obtained from the patient, thereby predicting the post-relapse survival of the patient, wherein the group of multiple myeloma genes comprises all of AMPD1, ANPEP, BIRC3, BMP6, BTLA, CBFA2T3, CCNE2, CCR2, CCR7, CD44, CD48, CD82, CEP170, CHST11, CISH, CITED2, DUSP1, EPB41L3, FAM129B, FER1L4, FOS, FOSB, GLA, GPRC5D, HBEGF, HMOX1, ICAM1, IFI30, IL8, ISG20, JUN, KANK1, KLF2, KLHL21, LIME1, LRRN1, MAFF, MMP14, MT1E, MT1H, PECAM1, PLAC8, PLAU, PLAUR, PRRG4, SAT1, SGK1, SIX5, SLC25A19, SLC2A4RG, SOCS3, SPHK1, TGM2, TMEM49, TNFAIP2, TNFRSF17, TSC22D3, and UGT2B17.
 11. The method of claim 1, wherein the plasma cells are CD138+.
 12. The method of claim 1, wherein testing the level of gene expression of the genes in the group of multiple myeloma genes comprises: hybridizing nucleic acids obtained from the multiple myeloma plasma cells to one or more platforms comprising probe sets hybridizable to one or more genes in the group of multiple myeloma genes; and converting intensity of a signal generated upon hybridization to the value of gene expression for each multiple myeloma gene in the group.
 13. The method of claim 1, wherein the level of gene expression is measured using an oligonucleotide array.
 14. The method of claim 13, wherein the oligonucleotide array is a U133Plus2.0 microarray.
 15. The method of claim 1, wherein the level of gene expression is measured by polymerase chain reaction (including quantitative PCR, real-time PCR, and quantitative real-time PCR (qRT-PCR)) or sequencing.
 16. A method of treating a relapsed multiple myeloma patient, comprising administering a suitable multiple myeloma therapy to patient evaluated by claim
 1. 17. The method of claim 16, wherein the patient is predicted to have a low rate of survival and the multiple myeloma therapy administered to the patient is a salvage therapy.
 18. (canceled) 